An "A-B" model has been proposed for several toxic peptides that cross cell membranes; any of these peptides, including cholera, pertussis, and diphtheria toxins, consists of two functionally distinct parts, an A component that is active enzymatically and a B component that binds to surface receptors to enable the A component to enter the cell where it acts. The interaction of these toxins with intact cells is characterized by a lag time that precedes the onset of their action, probably a time required for the A component to traverse the plasma membranes. In the case of IAP, there was a definite lag period of one hour before alpha-adrenergic inhibition of insulin secretion began to be reversed progressively in islet cultures with isletactivating protein, IAP.
Pertussis toxin, an exotoxin produced by Bordetella pertussis, comprises these two components: an enzymatically active A subunit and a B oligomer which is responsible for binding of the toxin to eukaryotic cell surfaces. The B oligomer, composed of five subunits ranging in molecular weights from 23,000 to 9,300, exhibits certain activities. For example, the B oligomer agglutinates erythrocytes and stimulates mitosis of lymphocytes. The A subunit, a single polypeptide chain having a molecular weight of 28,000, catalyzes the ADP-ribosylation of a family of GTP-binding regulatory proteins found in eukaryotic cells. In the absence of a protein substrate, the A subunit will catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. The ADP-ribosyltransferase activity of the toxin is believed to be responsible for a number of biological effects observed both in vivo and in vitro. For example, ADP-ribosylation of the GTP-binding protein termed G.sub.i can result in interference with the ability of the cell to respond to hormones which inhibit cyclic AMP production or mobilize calcium.
Pertussis toxin resembls cholera toxin and diphtheria toxin in that it comprises two types of components, one enzymatically active and the other responsible for the binding of the toxin to the eukaryotic cell surface and the introduction of the active subunit into the cell. The holotoxin and the isolated A subunit have been described to be equally effective on a molar basis in ADP-ribosylating G.sub.i in crude membrane preparations from certain cell types such as C6 glioma cells. However, only the subunit and not the holotoxin was reported to be effective in transferring the ADP-ribose moiety of NAD to G.sub.i in crude membrane preparations of rabbit platelets and rat mast cells. Moreover, the A subunit is more active than the holotoxin on a molar basis in catalyzing the hydrolytic cleavage of NAD to ADP-ribose and nicotinamide in vitro.
Tamura et al. in Biochemistry 21;5516-5522, 1982, describe the analysis of the B oligomer of pertussis toxin by degrading the toxin with sodium dodecyl sulfate followed by gel electrophoresis. The subunits were separated by exposure of the material to 5M ice-cold urea for four days, followed by column chromatography with carboxymethyl-Sepharose. This yielded sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, S-2 and S-4 for one dimer and S-3 and S-4 for the other, after sixteen hours of exposure to 8 M urea. These subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native toxin in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats.
In Japanese patent No. 59110626 is described a pertussis vaccine containing, as the active ingredient, the B oligomer of pertussis toxin. Aluminum phosphate or aluminum hydroxide is incorporated as an adjuvant. The new vaccine disclosed is free from side effects and does not substantially contain any endotoxin, and has a higher phylaxis effect.
Helting, in U.S. Pat. No. 4,029,766, discloses a method for making a protective antigen from Bordetella pertussis by mixing the pathogens with an aqueous solution of a denaturing agent and a neutral salt, separating the liquid supernatant containing the protective antigen suspended therein from the residue, and subsequently separating the denaturing agent from the aqueous suspension of the protective antigen.
Tint, in U.S. Pat. No. 2,772,201, discloses a method for fractionating and concentrating proteins of bacterial origin, such as from pertussis, by precipitation of all of the protein complex molecules having an isoelectric pH equal to or higher than that of the active protein, separating them from the solution, and fractionating them.
Ribi et al., in U.S. Pat. No. 4,435,386, and Ribi in U.S. Pat. Nos. 4,436,727 and 4,436,728, disclose a method of making a refined detoxified endotoxin product by reacting endotoxic extract with an inorganic or organic acid and lyophilizing to produce a hydrolyzed crude lipid A. This product is treated with a solvent to dissolve out impurities.
In Japanese Patent No. 53222032, there is described the use of pertussis subunits S2, S3, and S4 in a vaccine.